Abstract

Retinoblastoma is the most common intraocular cancer in children. While the primary tumor can often be treated by local or systemic chemotherapy, metastatic dissemination is generally resistant to therapy and remains a leading cause of pediatric cancer death in much of the world. In order to identify new therapeutic targets in aggressive tumors, we sequenced RNA transcripts in five snap frozen retinoblastomas which invaded the optic nerve and five which did not. A three-fold increase was noted in mRNA levels of ACVR1C/ALK7, a type I receptor of the TGF-β family, in invasive retinoblastomas, while downregulation of DACT2 and LEFTY2, negative modulators of the ACVR1C signaling, was observed in most invasive tumors. A two- to three-fold increase in ACVR1C mRNA was also found in invasive WERI Rb1 and Y79 cells as compared to non-invasive cells in vitro. Transcripts of ACVR1C receptor and its ligands (Nodal, Activin A/B, and GDF3) were expressed in six retinoblastoma lines, and evidence of downstream SMAD2 signaling was present in all these lines. Pharmacological inhibition of ACVR1C signaling using SB505124, or genetic downregulation of the receptor using shRNA potently suppressed invasion, growth, survival, and reduced the protein levels of the mesenchymal markers ZEB1 and Snail. The inhibitory effects on invasion, growth, and proliferation were recapitulated by knocking down SMAD2, but not SMAD3. Finally, in an orthotopic zebrafish model of retinoblastoma, a 55% decrease in tumor spread was noted (p = 0.0026) when larvae were treated with 3 µM of SB505124, as compared to DMSO. Similarly, knockdown of ACVR1C in injected tumor cells using shRNA also resulted in a 54% reduction in tumor dissemination in the zebrafish eye as compared to scrambled shRNA control (p = 0.0005). Our data support a role for the ACVR1C/SMAD2 pathway in promoting invasion and growth of retinoblastoma.

a-c: Representative images of the MRI evaluation of the optic nerve invasion in the retinoblastoma cases. a: Axial T1 WI post contrast fat suppression of case #2 shows bilateral retinoblastoma with clear delineation of a continuous choroidal-retinal line with no sign of optic nerve invasion (arrow). b: Axial T1-weighted post contrast, fat-suppressed image of case #8: retinoblastoma with post laminar optic nerve invasion (arrowhead). c: Axial post contrast T1WI, fat-suppressed image of case #9: retinoblastoma which diffusely infiltrated the sclera and the optic nerve. d: The mRNA levels of ACVR1C were significantly increased in all five invasive cases, while those of DACT2 and LEFTY2 were significantly decreased in most of the invasive cases. Expression levels were obtained from the RNA-seq analysis. Probability of differential expression: PPDE=0.814 (ACVR1C); PPDE=0.995 (DACT2); PPDE=0.892 (LEFTY2). e: ACVR1C mRNA levels were significantly increased in invasive WERI Rb1 and Y79 cells, as compared to non-invasive cells. Transwell invasion assay was used to separate the invasive cells, present in the lower side of a Matrigel-coated filter, from the non-invasive, present inside of the insert, after 72 hours of incubation, following a previous procedure ().

The mRNA levels of ACVR1C receptor (a) and ligands (b-e) in multiple retinoblastoma lines were determined by qPCR. P values were calculated using one-way analysis of the variance (ANOVA), with post-hoc Tukey’s test in the table. Levels of phospho- and total SMAD2/3 proteins, Nodal and GDF3 ligands in retinoblastoma lines were evaluated by Western blot (f), using β-Actin as loading control. Pancreatic cancer cell line PANC-1, treated with TGF-β1 at 10 ng/mL for 2 hours, was used as positive control for phospho-SMAD3 antibody. Nodal expression was determined in WERI Rb1 and Y79 cells by immunofluorescence (g), using anti-Nodal antibody (red). Nuclei were stained with DAPI (blue). PANC-1 cells were used as positive control.

Pharmacological inhibition of the ACVR1C/SMAD2 pathway using SB505124 represses growth and invasion in retinoblastoma cells.

a, c, e: Growth was inhibited in a dose-dependent manner in WERI Rb1 (a), Y79 (c), and HSJD-RBVS-10 (e) cells treated with SB505124 for 3, 5, 7 days at the indicated doses, as found by CCK-8 assay. P values were calculated using two-sided Student t-test vs DMSO-treated cells. Data are presented as the mean + SD. b, d, f: The ability of the cells to invade a Matrigel-coated filter was reduced in a dose-dependent manner in WERI Rb1 (b), Y79 (d), and HSJD-RBVS-10 (f) cells treated with SB505124 for 3 days at the indicated doses, as found by transwell invasion assay.

Pharmacological inhibition of the ACVR1C/SMAD2 pathway induces apoptosis and inhibits the expression of EMT markers in retinoblastoma cells.

a-c: Phosphorylation of SMAD2 was reduced in a dose-dependent manner in WERI Rb1 (a), Y79 (b), HSJD-RBVS-10 (c) cells treated with SB505124 for 4 days at the indicated doses, as found by Western blot. Induction of cleaved PARP, indicative of apoptosis, and reduction in Snail and ZEB1 protein levels were also observed, starting at the dose of 2 μM. No phosphorylation of SMAD3 was detected in these lines, while a dose-dependent decrease in the protein levels of SMAD3 was observed in WERI Rb1 and Y79 (, bottom panel). PANC-1 cells treated with TGF-β1 at 10 ng/mL for 2 hours were used as positive control for phospho-SMAD3 antibody. d,e: Treatment with SB505124 for 3 days significantly increased apoptosis at 4 and 8 μM in WERI Rb1 (d) and at 2, 4, 8 μM in Y79 (e), compared to DMSO, as determined by immunofluorescence assay using an antibody specific for cleaved caspase-3 (red). P values were calculated using two-sided Student t-test vs DMSO-treated cells. Data are presented as mean + SD. Microphotographs in the right part of the panels are representative images of the immunofluorescence staining. Nuclei were stained with DAPI (blue).

ACVR1C (a) and Snail (c) mRNA levels were determined by qPCR in Y79-GFP cells transduced with ACVR1C shRNAs or scrambled shRNA, and in parental cells. Invasion was reduced by about 70% in Y79-GFP cells expressing ACVR1C shRNAs compared to scrambled shRNA, as determined by transwell invasion assay (b). P values were calculated using two-sided Student t-test vs scrambled shRNA. Data are presented as mean + SD. Phosphorylation of SMAD2 and protein levels of Snail and ZEB1 were dramatically reduced in cells expressing ACVR1C shRNAs as compared to scrambled shRNA, while the apoptotic marker cleaved PARP was increased, as found by Western blot (d). Growth was reduced by more than 90% in Y79 cells expressing two different ACVR1C shRNAs, compared to scrambled shRNA, as found by CCK-8 growth assay (e). The percentage of Ki67-positive cells was reduced from 40 to 50% in cells transduced with two different ACVR1C shRNAs, compared to scrambled shRNA, as found by Ki67 proliferation assay (f).

a: Representative images of the localization of the Y79-GFP cells (green dots), as they were monitored by confocal intravital microscopy at 1 and 4 days post-injection (dpi). The red sphere represents the minimum bounding sphere (MBS), indicative of the dissemination of the cells outside the injection point (50 μm grid for scale). The diffuse green autofluorescence, more evident at 4 dpi, is due to the endogenous iridophore pigmentation of the zebrafish, which was accounted for during the MBS analysis. b: The MBS diameter (μm) was significantly increased from day 1 to day 4 when zebrafish larvae (n=12) were treated with DMSO for 4 days, upon injection of Y79-GFP cells in the vitreous cavity, as opposed to treatment with SB505124 (3 μM), which did not produce any significant increase in the MBS diameter. Effect size for DMSO treatment: 106.91; 95% confidence interval (CI): 33.64, 179.99; p=0.0082; effect size for SB505124 treatment: 6.75; 95% CI:−18.96, 32.46; p=0.59. c: 55% reduction in the fold-change of the MBS diameter at 4dpi/1dpi was observed when larvae were treated with 3 μM of SB505124 compared to DMSO. Effect size: −0.57; 95% CI: −0.91, −0.25; p=0.0026. The extent of retinoblastoma dissemination, represented by the MBS diameter, was determined using IMARIS & Matlab software. 50 μm grid for scale.